Structure elucidation of dimeric transmembrane domains of bitopic proteins

Abstract

The interaction between transmembrane helices is of great interest because it directly determines biological activity of a membrane protein. Either destroying or enhancing such interactions can result in many diseases related to dysfunction of different tissues in human body. One much studied form of membrane proteins known as bitopic protein is a dimer containing two membrane-spanning helices associating laterally. Establishing structure-function relationship as well as rational design of new types of drugs targeting membrane proteins requires precise structural information about this class of objects. At present time, to investigate spatial structure and internal dynamics of such transmembrane helical dimers, several strategies were developed based mainly on a combination of NMR spectroscopy, optical spectroscopy, protein engineering and molecular modeling. These approaches were successfully applied to homo- and heterodimeric transmembrane fragments of several bitopic proteins, which play important roles in normal and in pathological conditions of human organism.

Figure 1

Spatial structure elucidation of dimeric TM domain of bitopic protein (exemplified by proapoptotic protein BNip3) using computer simulations techniques. (A) Collection of rough models via Monte Carlo conformational search in implicit membranes. Set of possible structure candidates having minimal energy is presented. Peptides are given in ribbon and stick representations. Positions of implicit membrane are selected by gray hatching. (B) Results of MD-relaxation of the MC structures in full-atom hydrated bilayer. Only structures stable in the membrane during MD are selected. Membranes are delineated by position of phosphorus atoms (shown as spheres). Other details were skipped for clarity. (C) The resulting structure selected via comparison with experimental information about dimer interface.

Figures 2

Spatial structure elucidation of dimeric TM domain of the bitopic protein (exemplified by receptor tyrosine kinase ErbB2) using with the aid of heteronuclear solution NMR in lipid bicelles and MD-relaxation in explicit lipid bilayer. (A) Acquisition of NMR spectra of isotopic “heterodimer,” consisting of ¹³C/¹⁵N-isotope labeled and natural abundance ErbB2 TM fragments (residues 641–685) embedded into DMPC/DHPC lipid bicelles. From left to right, ¹H-¹⁵N HSQC spectrum with amide backbone resonance assignments, two representative 2D strips from the 3D ¹³C F1-filtered/F3-edited-NOESY spectrum with intermolecular protein-protein and protein-lipid NOE contacts are presented. (B) Determination of high-resolution spatial structure of the right-handed ErbB2 TM homodimer in lipid bicelle using NMR-derived restraints. The obtained N-terminal association mode of the ErbB2 TM dimer via N-terminal dimerization motif corresponds to the receptor active state. (C) MD-relaxation of the ErbB2 TM homodimer in hydrated explicit DMPC lipid bilayer with imposed NMR-derived constraints. Yellow balls show phosphorus atoms of lipid heads. The spatial locations of the three characteristic dimerization motifs of ErbB2tm are marked by dashed ovals. (D) Analysis of interacting surfaces of the ErbB2 TM helices. In left, hydrophobic and hydrophilic (polar) surfaces of one TM helix in the homodimer colored in yellow and green according to the molecular hydrophobicity potential (MHP). The second monomer of the dimer is shown with red side chains. In right, hydrophobicity map for ErbB2 TM helix surface with contour isolines encircling hydrophobic regions with high values of MHP is presented with red-point area indicating the helix packing interface via N-terminal glycine zipper motif T⁶⁵²xxxS⁶⁵⁶xxxG⁶⁶⁰. The residues composing C-terminal unemployed dimerization GG4-like motif G⁶⁶⁸xxxG⁶⁷² are highlighted in green. (E) Local structure analysis of intra and intermolecular interactions in the ErbB2 TM dimer. Comparison of intermonomeric hydrogen bonding (black dotted lines) in the TM helix-helix interface of ErbB2 and its constitutively active Val659Glu-mutant is presented.